K63-Linked Ubiquitination Targets Toxoplasma gondii for Endo-lysosomal Destruction in IFNγ-Stimulated Human Cells

Abstract

Toxoplasma gondii is the most common protozoan parasitic infection in man. Gamma interferon (IFNγ) activates haematopoietic and non-haematopoietic cells to kill the parasite and mediate host resistance. IFNγ-driven host resistance pathways and parasitic virulence factors are well described in mice, but a detailed understanding of pathways that kill Toxoplasma in human cells is lacking. Here we show, that contrary to the widely held belief that the Toxoplasma vacuole is non-fusogenic, in an immune-stimulated environment, the vacuole of type II Toxoplasma in human cells is able to fuse with the host endo-lysosomal machinery leading to parasite death by acidification. Similar to murine cells, we find that type II, but not type I Toxoplasma vacuoles are targeted by K63-linked ubiquitin in an IFNγ-dependent manner in non-haematopoetic primary-like human endothelial cells. Host defence proteins p62 and NDP52 are subsequently recruited to the type II vacuole in distinct, overlapping microdomains with a loss of IFNγ-dependent restriction in p62 knocked down cells. Autophagy proteins Atg16L1, GABARAP and LC3B are recruited to <10% of parasite vacuoles and show no parasite strain preference, which is consistent with inhibition and enhancement of autophagy showing no effect on parasite replication. We demonstrate that this differs from HeLa human epithelial cells, where type II Toxoplasma are restricted by non-canonical autophagy leading to growth stunting that is independent of lysosomal acidification. In contrast to mouse cells, human vacuoles do not break. In HUVEC, the ubiquitinated vacuoles are targeted for destruction in acidified LAMP1-positive endo-lysosomal compartments. Consequently, parasite death can be prevented by inhibiting host ubiquitination and endosomal acidification. Thus, K63-linked ubiquitin recognition leading to vacuolar endo-lysosomal fusion and acidification is an important, novel virulence-driven Toxoplasma human host defence pathway.

Visualizing ubiquitination in mammalian cells

“…SRM confirmed non-uniform distributions of several of these ubiquitin structures within the coat in which some chain-linkages accumulated in micro domain-like structures [92,138]. In addition, SIM was also used to image the distribution of the autophagic adaptor proteins p62 and NDP52 on the type II Toxoplasma gondii parasitophorous vacuole [140]. Quantitative dSTORM imaging of M1 signals in the Salmonella Ub coat revealed striking alterations in M1 antibody distribution and intensity in infected OTULIN-depleted cells, without affecting K63 Ub, suggesting OTULIN as crucial regulator of linear chain deposition within the ubiquitin coat ( Fig 4A and B) [138].…”

“…For example, immunofluorescence staining of Salmonella ‐infected human epithelial cells with FK1, FK2 and chain‐specific antibodies and imaging of the recruitment of GFP‐tagged linear and K63‐specific UBDs expressed in infected cells confirmed the presence of linear, K11‐, K63‐ and K48‐linked chains around the cytosolic Salmonella . These approaches contributed to the identification and characterization of key E3 ligases, like LUBAC , PARKIN , LRSAM1 and ARIH1 , of the DUB OTULIN and several UBD proteins that recruit the autophagy machinery (like SQSTM1/p62, NDP52 and OPTN) . In addition, multiscale imaging has been applied to explore the role of Ub in xenophagy of other intracellular microorganisms, like Shigella and Mycobacterium and even internalized latex beads that mimic cytosolic bacteria .…”

ToxoplasmaGRA15 limits parasite growth in IFNγ-activated fibroblasts through TRAF ubiquitin ligases

“…Toxoplasma strain-dependent susceptibility to IFNγ in different human and murine cells in vitro is well established (Bando et al, 2018;Clough et al, 2016;Haldar et al, 2015;Niedelman et al, 2012;Qin et al, 2017;Selleck et al, 2013Selleck et al, , 2015Niedelman et al, 2013). However, Toxoplasma effectors that affect strain differences in susceptibility to IFNγ-induced cell autonomous immunity in human cells have not been described.…”

Mimicry embedding for advanced neural network training of 3D biomedical micrographs

“…croscopy, detecting and quantifying intracellular viability at the single parasite level is challenging (13). To generate a Tg viability training dataset, cells infected with Tg-EGFP (c1) were fixed and stained with fluorescent markers of DNA (c2), and host cell ubiquitin (c3) which was used a weak label to annotate the subset of "unviable" parasites (13,29) (Fig. 5a).…”